Porous particles comprising excipients for deep lung delivery

ABSTRACT

Improved porous particles for drug delivery to the pulmonary system, and methods for their synthesis and administration are provided. In a preferred embodiment, the porous particles are made of a biodegradable material and have a mass density less than 0.4 g/cm 3 /. The particles may be formed of biodegradable materials such as biodegradable polymers. For example, the particles may be formed of a functionalized polyester graft copolymer consisting of a linear a-hydroxy-acid polyester backbone having at least one amino acid group incorporated therein and at least one poly(amino acid) side chain extending from an amino acid group in the polyester backbone. In one embodiment, porous particles having a relatively large mean diameter, for example greater than 5 μm, can be used for enhanced delivery of a therapeutic agent to the alveolar region of the lung. The porous particles incorporating a therapeutic agent may be effectively aerosolized for administration to the respiratory tract to permit systemic or local delivery of wide variety of therapeutic agents.

RELATED APPLICATIONS

This application is a Continuation of U.S application Ser. No.10/209,654, filed on Jul. 30, 2002 now U.S. Pat No. 6,740,310, which isa Continuation of U.S. application Ser. No. 09/888,688 filed on Jun. 25,2001 now U.S. Pat. No. 6,436,443, which is a Continuation of U.S.application Ser. No. 09/569,153 filed on May 11, 2000, now U.S. Pat. No.6,254,854, which is a Continuation of U.S. application Ser. No.08/655,570, filed May 24, 1996, now abandoned; the entire teachings ofall referenced applications and patents are incorporated herein byreference.

BACKGROUND OF THE INVENTION

The present invention relates generally to porous polymeric particlesfor drug delivery to the pulmonary system.

Biodegradable polymeric particles have been developed for thecontrolled-release and delivery of protein and peptides drugs. Langer,R., Science, 249: 527-1533 (1990). Examples include the use ofbiodegradable particles for gene therapy (Mulligan, R. C., Science,260:926-932 (1993)) and “single-shot” vaccine delivery (Eldridge et al.,Mol. Immunol., 28:287-294 (1991)) for immunization. Protein and peptidedelivery via degradable particles is restricted due to lowbioavailability in the blood stream, since macromolecules and/ormicroparticles tend to poorly permeate organ-blood barriers of the humanbody, particularly when delivered either orally or invasively.

Aerosols for the delivery of therapeutic agents to the respiratory tracthave been developed. The respiratory tract includes the upper airways,including the oropharynx

Aerosols for the delivery of therapeutic agents to the respiratory tracthave been developed. The respiratory tract includes the upper airways,including the oropharynx and larynx, followed by the lower airways,which include the trachea followed by bifurcations into the bronchi andbronchioli. The upper and lower airways are called the conductiveairways. The terminal bronchioli then divide into respiratory bronchioliwhich then lead to the ultimate respiratory zone, the alveoli, or deeplung. Gonda, I. “Aerosols for delivery of therapeutic and diagnosticagents to the respiratory tract,” In Critical Reviews in TherapeuticDrug Carrier Systems, 6:273-313, (1990). The deep lung, or alveoli, arethe primary target of inhaled therapeutic aerosols for systemicdelivery.

Inhaled aerosols have been used for the treatment of local lungdisorders including asthma and cystic fibrosis (Anderson, et al., Am.Rev. Rev. Respir. Dis., 140:1317-1324 (1989)) and have potential for thesystemic delivery of peptides and proteins as well (Patton and Platz,Advanced Drug Delivery Reviews, 8:179-196 (1992)). However, pulmonarydrug delivery strategies present many difficulties for the delivery ofmacromolecules; these include protein denaturation duringaerosolization, excessive loss of inhaled drug in the oropharyngealcavity (typically exceeding 80%), poor control over the site ofdeposition, irreproducibility of therapeutic results owing to variationsin breathing patterns, the quick absorption of drug potentiallyresulting in local toxic effects, and phagocytosis by lung macrophages.

Local and systemic inhalation therapies can often benefit from arelative slow controlled release of the therapeutic agent. Gonda, I.,“Physico-chemical principles in aerosol delivery,” In Topics inPharmaceutical Science, 1991, D. J. A. Crommelin and K. K. Midha, Eds.,Stuttgart: Medpharm Scientific Publishers, pp. 95-117, 1992. Slowrelease from a therapeutic aerosol can prolong the residence of anadministered drug in the airways or acini, and diminish the rate of drugappearance in the blood stream. Also, patient compliance is increased byreducing the frequency of dosing. Langer, R., Science, 249:1527-1533(1990); and Gonda, I., “Aerosols for delivery of therapeutic anddiagnostic agents to the respiratory tract,” In Critical Reviews inTherapeutic Drug Carrier Systems, 6:273-313, 1990.

The human lungs can remove or rapidly degrade hydrolytically cleavabledeposited aerosols over periods ranging from minutes to hours. In theupper airways, ciliated epithelia contribute to the “mucociliaryexcalator” by which particles are swept from the airways toward themouth. Pavia, D., “LungMucociliary Clearance,” In Aerosols and the Lung:Clinical and Experimental Aspects, Clarke, S. W. and Pavia, D., Eds.,Butterworths, London, 1984. In the deep lungs, alveolar macrophages arecapable of phagocytosing particles soon after their deposition. Warheit,M. B. and Hartsky, M. A., Microscopy Res. Tech., 26:412-422 (1993); andBrain, J. D., “Physiology and Pathophysiology of Pulmonary Macrophages,”In The Reticuloendothelial System, S. M. Reichard and J. Filkins, Eds.,Plenum, New York, pp. 315-327, 1985. As the diameter of particlesexceeds 3 μm, there is increasingly less phagocytosis by macrophages.However, increasing the particle size also minimizes the probability ofparticles (possessing standard mass density) entering the airways andacini due to excessive deposition in the oropharyngeal or nasal regions.Heyder, J., et al., J. Aerosol Sci., 17: 811-825 (1986). An effectiveslow-release inhalation therapy requires a means of avoiding orsuspending the lung's natural clearance mechanisms until drugs have beeneffectively delivered.

Therapeutic dry-powder aerosols have been made as solid (macroscopicallynonporous) particles, with mean diameters less than approximately 5 μmto avoid excessive oropharyngeal deposition. Ganderton, D., J.Biopharmaceutical Sciences, 3:101-105 (1992); and Gonda, I.,“Physico-Chemical Principles in Aerosol Delivery,” In Topics inPharmaceutical Sciences, 1991, Commelin, D. J. and K. K. Midha, Eds.,Medpharm Scientific Publishers, Stuttgart, pp. 95-115, 1992.

There is a need for improved inhaled aerosols for pulmonary delivery oftherapeutic agents. There is a need for the development of drug carrierswhich are capable of delivering the drug in an effective amount into theairways or the alveolar zone of the lung. There further is a need forthe development of drug carriers for use as inhaled aerosols which arebiodegradable and are capable of controlled release within the airwaysor in the alveolar zone of the lung.

It is therefore an object of the present invention to provide improvedcarriers for the pulmonary delivery of therapeutic agents. It is afurther object of the invention to provide inhaled aerosols which areeffective carriers for delivery of therapeutic agents to the deep lung.It is another object of the invention to provide carriers for pulmonarydelivery which avoid phagocytosis in the deep lung. It is a furtherobject of the invention to provide carriers for pulmonary drug deliverywhich are capable of biodegrading and releasing the drug at a controlledrate.

SUMMARY OF THE INVENTION

Improved porous particles for drug delivery to the pulmonary system, andmethods for their synthesis and administration are provided. In apreferred embodiment, the porous particles are made of a biodegradablematerial and have a mass density less than 0.4 g/cm³. The particles maybe formed of biodegradable materials such as biodegradable polymers. Forexample, the particles may be formed of a functionalized polyester graftcopolymer consisting of a linear .alpha.-hydroxy-acid polyester backbonehaving at least one amino acid group incorporated therein and at leastone poly(amino acid) side chain extending from an amino acid group inthe polyester backbone. In one embodiment, porous particles having arelatively large mean diameter, for example, greater than 5 μm, can beused for enhanced delivery of a therapeutic agent to the airways or thealveolar region of the lung. The porous particles incorporating atherapeutic agent may be effectively aerosolized for administration tothe respiratory tract to permit systemic or local delivery of a widevariety of therapeutic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph comparing total particle mass of porous and non-porousparticles deposited on the nonrespirable and respirable stages of acascade impactor following aerosolization.

FIG. 2 is a graph comparing total particle mass deposited in the tracheaand after the carina (lungs) in rat lungs and upper airways followingintratracheal aerosolization during forced ventilation of porousPLAL-Lys particles and non-porous PLAL particles.

FIG. 3 is a graph comparing total particle recovery of porous PLAL-Lysparticles and non-porous PLAL particles in rat lungs and followingbroncho alveolar lavage.

DETAILED DESCRIPTION OF THE INVENTION

Biodegradable particles for improved delivery of therapeutic agents tothe respiratory tract are provided. The particles can be used in oneembodiment for controlled systemic or local drug delivery to therespiratory tract via aerosolization. In one embodiment, the particlesare porous particles having a mass density less than 1.0 g/cm³,preferably less than about 0.4 g/cm³. The porous structure permits deeplung delivery of relatively large diameter therapeutic aerosols, forexample greater than 5 μm in mean diameter. The particles also mayinclude a rough surface texture which can reduce particle agglomerationand provide a highly flowable powder, which is ideal for aerosolizationvia dry powder inhaler devices, leading to lower deposition in the mouthand throat.

Mass Density and Diameter of Porous Particles

As used herein the term “porous particles” refers to particles having atotal mass density less than about 0.4 g/cm³. The mean diameter of theparticles can range, for example, from about 100 nm to 15 μm, or largerdepending on factors such as particle composition, and the targeted siteof the respiratory tract for deposition of the particle.

Particle Size

In one embodiment, particles which are macroscopically porous, andincorporate a therapeutic drug, and having a mass density less thanabout 0.4 g/cm³, can be made with mean diameters greater than 5 μm, suchthat they are capable of escaping inertial and gravitational depositionin the oropharyngeal region, and are targeted to the airways or the deeplung. The use of larger porous particles is advantageous since they areable to aerosolize more efficiently than smaller, non-porous aerosolssuch as those currently used for inhalation therapies.

The large (>5 μm) porous particles are also advantageous in that theycan more successfully avoid phagocytic engulfment by alveolarmacrophages and clearance from the lungs, in comparison to smallernon-porous particles, due to size exclusion of the particles from thephagocytes' cytosolic space. Phagocytosis of particles by alveolarmacrophages diminishes precipitously as particle diameter increasesbeyond 3 μm. Kawaguchi, H. et al., Biomaterials, 7:61-66 (1986); Krenis,L. J. and Strauss, B., Proc. Soc. Exp. Med., 107:748-750 (1961); andRudt, S. and Muller, R. H., J. Contr. Rel., 22:263-272 (1992). Theporous particles thus are capable of a longer term release of atherapeutic agent. Following inhalation, porous degradable particles candeposit in the lungs (due to their relatively low mass density), andsubsequently undergo slow degradation and drug release, without themajority of the particles being phagocytosed by alveolar macrophages.The drug can be delivered relatively slowly into the alveolar fluid, andat a controlled rate into the blood stream, minimizing possible toxicresponses of exposed cells to an excessively high concentration of thedrug. The porous polymeric particles thus are highly suitable forinhalation therapies, particularly in controlled release applications.The preferred diameter for porous particles for inhalation therapy isgreater than 5 μm, for example between about 5-15 μm.

The particles also may be fashioned with the appropriate material,diameter and mass density for localized delivery to other regions of therespiratory tract such as the upper airways. For example higher densityor larger particles may be used for upper airway delivery.

Particle Deposition

Inertial impaction and gravitational settling of aerosols arepredominant deposition mechanisms in the airways and acini of the lungsduring normal breathing conditions. Edwards, D. A., J. Aerosol Sci.,26:293-317 (1995). Both deposition mechanisms increase in proportion tothe mass of aerosols and not to particle volume. Since the site ofaerosol deposition in the lungs is determined by the intrinsic mass ofthe aerosol (at least for particles of mean aerodynamic diameter greaterthan approximately 1 μm), diminishing particle mass density byincreasing particle porosity permits the delivery of larger particlesinto the lungs, all other physical parameters being equal.

The low mass porous particles have a small aerodynamic diameter incomparison to the actual sphere diameter. The aerodynamic diameter,d_(ae), is related to the actual sphere diameter, d (Gonda, I.,“Physico-chemical principles in aerosol delivery,” In Topics inPharmaceutical Sciences, 1991, (eds. D. J. A. Crommelin and K. K.Midha), pp. 95-117, Stuttgart: Medpharm Scientific Publishers, 1992) bythe formula:d_(aer)=d_(√ρ)where the particle mass density ρ is in units of g/cm³. Maximaldeposition of monodisperse aerosol particles in the alveolar region ofthe human lung (˜60%) occurs for an aerodynamic diameter ofapproximately d_(aer)=3 μm. Heyder, J. et al., J. Aerosol Sci., 17:811-825 (1986). Due to their small mass density, the actual diameter dof porous particles comprising a mondisperse inhaled powder that willexhibit maximum deep-lung deposition is:d=3/√ρμm (where ρ<1);where d is always greater than 3 μm. For example, porous particles thatdisplay a mass density, ρ=0.1 g/cm³, will exhibit a maximum depositionfor particles having actual diameters as large as 9.5 μm. The increasedparticle size diminishes interparticle adhesion forces. Visser, J.,Powder Technology, 58:1-10. Thus, large particle size increasesefficiency of aerosolization to the deep lung for particles of low massdensity.Particle Materials

The porous particles preferably arc biodegradable and biocompatible, andoptionally are capable of biodegrading at a controlled rate for deliveryof a drug. The porous particles can be made of any material which iscapable of forming a porous particle having a mass density less thanabout 0.4 g/cm³. Both inorganic and organic materials can be used. Forexample, ceramics may be used. Other non-polymeric materials may be usedwhich are capable of forming porous particles as defined herein.

Polymeric Particles

The particles may be formed from any biocompatible, and preferablybiodegradable polymer, copolymer, or blend, which is capable of formingporous particles having a density less than about 0.4 g/cm³.

Surface eroding polymers such as polyanhydrides may be used to form theporous particles. For example, polyanhydrides such aspoly[(p-carboxyphenoxy)hexane anhydride] (“PCPH”) may be used.Biodegradable polyanhydrides are described, for example, in U.S. Pat.No. 4,857,311, the disclosure of which is incorporated herein byreference.

In another embodiment, bulk eroding polymers such as those based onpolyesters including poly(hydroxy acids) can be used. For example,polyglycolic acid (“PGA”) or polylactic acid (“PLA”) or copolymersthereof may be used to form the porous particles, wherein the polyesterhas incorporated therein a charged or functionalizable group such as anamino acid as described below.

Other polymers include polyamides, polycarbonates, polyalkylenes such aspolyethylene, polypropylene, poly(ethylene glycol), poly(ethyleneoxide), poly(ethylene terephthalate), polyvinyl compounds such aspolyvinyl alcohols, polyvinyl ethers, and polyvinyl esters, polymers ofacrylic and methacrylic acids, celluloses, polysaccharides, and peptidesor proteins, or copolymers or blends thereof which are capable offorming porous particles with a mass density less than about 0.4 g/cm³.Polymers may be selected with or modified to have the appropriatestability and degradation rates in vivo for different controlled drugdelivery applications.

Polyester Graft Copolymers

In one preferred embodiment, the porous particles are formed fromfunctionalized polyester graft copolymers, as described in Hrkach etal., Macromolecules, 28:4736-4739 (1995); and Hrkach et al.,“Poly(L-Lactic acid-co-amino acid) Graft Copolymers: A Class ofFunctional Biodegradable Biomaterials” In Hydrogel and BiodegradablePolymers for Bioapplications, ACS Symposium Series No. 627, Raphael M.Ottenbrite et al., Eds., American Chemical Society, Chapter 8, pp.93-101, 1996, the disclosures of which are incorporated herein byreference. The functionalized graft copolymers are copolymers ofpolyesters, such as poly(glycolic acid) or poly(lactic acid), andanother polymer including functionalizable or ionizable groups, such asa poly(amino acid). In a preferred embodiment, comb-like graftcopolymers are used which include a linear polyester backbone havingamino acids incorporated therein, and poly(amino acid) side chains whichextend from the amino acid groups in the polyester backbone. Thepolyesters may be polymers of α-hydroxy acids such as lactic acid,glycolic acid, hydroxybutyric acid and valeric acid, or derivatives orcombinations thereof. The inclusion of ionizable side chains, such aspolylysine, in the polymer has been found to enable the formation ofmore highly porous particles, using techniques for making microparticlesknown in the art, such as solvent evaporation. Other ionizable groups,such as amino or carboxyl groups, may be incorporated, covalently ornoncovalently, into the polymer to enhance porosity. For example,polyaniline could be incorporated into the polymer.

An exemplary polyester graft copolymer, which may be used to form porouspolymeric particles is the graft copolymer, poly(lacticacid-co-lysine-graft-lysine) (“PLAL-Lys”), which has a polyesterbackbone consisting of poly(L-lactic acid-co-Z-L-lysine) (PLAL), andgrafted lysine chains. PLAL-Lys is a comb-like graft copolymer having abackbone composition, for example, of 98 mol % lactic acid and 2 mol %lysine and poly(lysine) side chains extending from the lysine sites ofthe backbone.

PLAL-Lys may be synthesized as follows. First, the PLAL copolymerconsisting of L-lactic acid units and approximately 1-2%Nε-carbobenzoxy-L-lysine (Z-L-lysine) units is synthesized as describedin Barrera et al., J. Am. Chem. Soc., 115:11010 (1993). Removal of the Zprotecting groups of the randomly incorporated lysine groups in thepolymer chain of PLAL yields the free e-amine which can undergo furtherchemical modification. The use of the poly(lactic acid) copolymer isadvantageous since it biodegrades into lactic acid and lysine, which canbe processed by the body. The existing backbone lysine groups are usedas initiating sites for the growth of poly(amino acid) side chains.

The lysine ε-amine groups of linear poly(L-lactic acid-co-L-lysine)copolymers initiate the ring opening polymerization of an amino acidN-carboxyanhydride (NCA) to produce poly(L-lactic acid-co-amino acid)comb-like graft copolymers. In a preferred embodiment, NCAs aresynthesized by reacting the appropriate amino acid with triphosgene.Daly et al., Tetrahedron Lett., 29:5859 (1988). The advantage of usingtriphosgene over phosgene gas is that it is a solid material, andtherefore, safer and easier to handle. It also is soluble in THF andhexane so any excess is efficiently separated from the NCAs.

The ring opening polymerization of amino acid N-carboxyanhydrides (NCAs)is initiated by nucleophilic initiators such as amines, alcohols, andwater. The primary amino initiated ring opening polymerization of NCAsallows good control over the degree of polymerization when the monomerto initiator ratio (M/I) is less than 150. Kricheldorf, H. R. In Modelsof Biopolymers by Ring-Opening Polymerization, Penezek, S., Ed., CRCPress, Boca Raton, 1990, Chapter 1; Kricheldorf, H. R.,α-Aminoacid-N-Carboxy-Anhydrides and Related Heterocycles,Springer-Verlag, Berlin, 1987; and Imanishi, Y. In Ring-OpeningPolymerization, Ivin, K. J. and Saegusa, T., Eds., Elsevier, London,1984, Volume 2, Chapter 8. Methods for using lysine ε-amine groups aspolymeric initiators for NCA polymerizations are described in the art.Sela, M. et al., J. Am. Chem. Soc., 78:746 (1956).

In the reaction of an amino acid NCA with PLAL, the nucleophilic primaryε-amine of the lysine side chain attacks C-5 of the NCA leading to ringopening and formation of the amino acid amide along with the evolutionof CO₂. Propagation takes place via further attack of the amine group ofthe amino acid amides on subsequent NCA molecules. The degree ofpolymerization of the poly(amino acid) side chains, the correspondingamino acid content in the graft copolymers and their resulting physicaland chemical characteristics can be controlled by changing the M/I ratiofor the NCA polymerization—that is, changing the ratio of NCA to lysineε-amine groups of pLAL. Thus, in the synthesis, the length of thepoly(amino acid), such as poly(lysine), side chains and the total aminoacid content in the polymer may be designed and synthesized for aparticular application.

The poly(amino acid) side chains grafted onto or incorporated into thepolyester backbone can include any amino acid, such as aspartic acid,alanine or lysine, or mixtures thereof. The functional groups present inthe amino acid side chains, which can be chemically modified, includeamino, carboxylic acid, sulfide, guanidino, imidazole and hydroxylgroups. As used herein, the term “amino acid” includes natural andsynthetic amino acids and derivatives thereof. The polymers can beprepared with a range of amino acid side chain lengths, for example,about 10-100 or more amino acids, and with an overall amino acid contentof, for example, 7-72% or more depending on the reaction conditions. Thegrafting of poly(amino acids) from the pLAL backbone may be conducted ina solvent such as dioxane, DMF, or CH₂Cl₂ or mixtures thereof. In apreferred embodiment, the reaction is conducted at room temperature forabout 2-4 days in dioxane.

Alternatively, the porous particles for pulmonary drug delivery may beformed from polymers or blends of polymers with differentpolyester/amino acid backbones and grafted amino acid side chains. Forexample, poly(lactic acid-co-lysine-graft-alanine-lysine)(“PLAL-Ala-Lys”), or a blend of PLAL-Lys with poly(lacticacid-co-glycolic acid-block-ethylene oxide) (“PLGA-PEG”)(“PLAL-Lys-PLGA-PEG”) may be used.

In the synthesis, the graft copolymers may be tailored to optimizedifferent characteristic of the porous particle including: i)interactions between the agent to be delivered and the copolymer toprovide stabilization of the agent and retention of activity upondelivery; ii) rate of polymer degradation and, thereby, rate of drugrelease profiles; iii) surface characteristics and targetingcapabilities via chemical modification; and iv) particle porosity.

Formation of Porous Polymeric Particles

Porous polymeric particles may be prepared using single and doubleemulsion solvent evaporation, spray drying, solvent extraction and othermethods well known to those of ordinary skill in the art. Methodsdeveloped for making microspheres for drug delivery are described in theliterature, for example, as described by Mathiowitz and Langer, J.Controlled Release, 5:13-22 (1987); Mathiowitz, et al., ReactivePolymers, 6:275-283 (1987); and Mathiowitz, et al., J. Appl. PolymerSci. 35:755-774 (1988), the teachings of which are incorporated herein.The selection of the method depends on the polymer selection, the size,external morphology, and crystallinity that is desired, as described,for example, by Mathiowitz, et al., Scanning Microscopy, 4:329-340(1990); Mathiowitz, et al., J. Appl. Polymer Sci., 45:125-134 (1992);and Benita, et al., J. Pharm. Sci., 73:1721-1724 (1984), the teachingsof which are incorporated herein.

In solvent evaporation, described for example, in Mathiowitz, et al.,(1990), Benita, and U.S. Pat. No. 4,272,398 to Jaffe, the polymer isdissolved in a volatile organic solvent, such as methylene chloride.Several different polymer concentrations can be used, for example,between 0.05 and 0.20 g/ml. The drug, either in soluble form ordispersed as fine particles, is added to the polymer solution, and themixture is suspended in an aqueous phase that contains a surface activeagent such as poly(vinyl alcohol). The aqueous phase may be, forexample, a concentration of 1% poly (vinyl alcohol) w/v in distilledwater. The resulting emulsion is stirred until most of the organicsolvent evaporates, leaving solid microspheres, which may be washed withwater and dried overnight in a lyophilizer.

Microspheres with different sizes (1-1000 microns) and morphologies canobtained by this method which is useful for relatively stable polymerssuch as polyesters and polystryrene. However, labile polymers such aspolyanhydrides may degrade due to exposure to water. For these polymers,solvent removal may be preferred.

Solvent removal was primarily designed for use with polyanhydrides. Inthis method, the drug is dispersed or dissolved in a solution of aselected polymer in a volatile organic solvent like methylene chloride.The mixture is then suspended in oil, such as silicon oil, by stirring,to form an emulsion. Within 24 hours, the solvent diffuses into the oilphase and the emulsion droplets harden into solid polymer microspheres.Unlike solvent evaporation, this method can be used to make microspheresfrom polymers with high melting points and a wide range of molecularweights. Microspheres having a diameter for example between one and 300microns can be obtained with this procedure.

Targeting of Particles

Targeting molecules can be attached to the porous particles via reactivefunctional groups on the particles. For example, targeting molecules canbe attached to the amino acid groups of functionalized polyester graftcopolymer particles, such as PLAL-Lys particles. Targeting moleculespermit binding interaction of the particle with specific receptor sites,such as those within the lungs. The particles can be targeted byattachment of ligands which specifically or non-specifically bind toparticular targets. Exemplary targeting molecules include antibodies andfragments thereof including the variable regions, lectins, and hormonesor other organic molecules capable of specific binding for example toreceptors on the surfaces of the target cells.

Therapeutic Agents

Any of a variety of therapeutic, prophylactic or diagnostic agents canbe delivered. Examples include synthetic inorganic and organiccompounds, proteins and peptides, polysaccharides and other sugars,lipids, and nucleic acid sequences having therapeutic, prophylactic ordiagnostic activities. Nucleic acid sequences include genes, antisensemolecules which bind to complementary DNA to inhibit transcription, andribozymes. The agents to be incorporated can have a variety ofbiological activities, such as vasoactive agents, neuroactive agents,hormones, anticoaguulants, immunomodulating agents, cytotoxic agents,antibiotics, antivirals, antisense, antigens, and antibodies. In someinstances, the proteins may be antibodies or antigens which otherwisewould have to be administered by injection to elicit an appropriateresponse. Compounds with a wide range of molecular weight can beencapsulated, for example, between 100 and 500,000 grams per mole.

Proteins are defined as consisting of 100 amino acid residues or more;peptides are less than 100 amino acid residues. Unless otherwise stated,the term protein refers to both proteins and peptides. Examples includeinsulin and other hormones. Polysaccharides, such as heparin, can alsobe administered.

The porous polymeric aerosols are useful as carriers for a variety ofinhalation therapies. They can be used to encapsulate small and largedrugs, release encapsulated drugs over time periods ranging from hoursto months, and withstand extreme conditions during aerosolization orfollowing deposition in the lungs that might otherwise harm theencapsulated therapeutic.

The porous particles may include a therapeutic agent for local deliverywithin the lung, such as agents for the treatment of asthma, emphysema,or cystic fibrosis, or for systemic treatment. For example, genes forthe treatment of diseases such as cystic fibrosis can be administered.

Administration

The particles including a therapeutic agent may be administered alone orin any appropriate pharmaceutical carrier, such as a liquid, for examplesaline, or a powder, for administration to the respiratory system.

Aerosol dosage, formulations and delivery systems may be selected for aparticular therapeutic application, as described, for example in Gonda,I. “Aerosols for delivery of therapeutic and diagnostic agents to therespiratory tract,” In Critical Reviews in Therapeutic Drug CarrierSystems, 6:273-313 (1990), and in Moren, “Aerosol dosage forms andformulations,” In Aerosols in Medicine. Principles, Diagnosis andTherapy, Moren, et al., Eds., Esevier, Amsterdam, 1985, the disclosuresof which are incorporated herein by reference.

The greater efficiency of aerosolization by porous particles ofrelatively large size permits more drug to be delivered than is possiblewith the same mass of nonporous aerosols. The relative large size ofporous aerosols depositing in the deep lungs also minimizes potentialdrug losses caused by particle phagocytosis. The use of porous polymericaerosols as therapeutic carriers provides the benefits of biodegradablepolymers for controlled released in the lungs and long-time local actionor systemic bioavailability. Denaturation of macromolecular drugs can beminimized during aerosolization since macromolecules are contained andprotected within a polymeric shell. Coencapsulation of peptides withpeptidase-inhibitors can minimize peptide enzymatic degradation.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLE 1 Synthesis of Porous Poly[(p-carboxyphenoxy)-hexane anhydride](“PCPH”) Particles

Porous poly[(p-carboxyphenoxy)-hexane anbydride] (“PCPH”) particles weresynthesized as follows. 100 mg PCPH (MW-25,000) was dissolved in 3.0 mLmethylene chloride. To this clear solution was added 5.0 mL 1w/v aqueouspolyvinyl alcohol (PVA, MW .about.25,000, 88 mole % hydrolyzed)saturated with methylene chloride, and the mixture was vortexed (VORTEXGENIE® 2 vortexer from Fisher Scientific) at maximum speed for oneminute. The resulting milky-white emulsion was poured into a beakercontaining 95 mL 1% PVA and homogenized (Silverson Homogenizers) at 6000RPM for one minute using a 0.75 inch tip. After homogenization, themixture was stirred with a magnetic stirring bar and the methylenechloride quickly extracted from the polymer particles by adding 2 mLisopropyl alcohol. The mixture was continued to stir for 35 minutes toallow complete hardening of the microparticles. The hardened particleswere collected by centrifugation and washed several times with doubledistilled water. The particles were freeze dried to obtain afree-flowing powder void of clumps. Yield, 85-90%.

The mean diameter of this batch was 6.0 μm, however, particles with meandiameters ranging from a few hundred nanometers to several millimetersmay be made with only slight modifications. Scanning electron micrographphotos of a typical barch of PCPH particles showed the particles to behighly porous. The particles have a mass density less that 1 g/cm³ asindicated by the fact that the particles float when dispersed in anorganic solvent.

EXAMPLE 2 Synthesis of PLAL-Lys and PLAL-Lys-Ala Polymeric andCopolymeric Particles

Porous PLAL-Lys Particles

PLAL-Lya particles were prepared by dissolving 50 mg of the graftcopolymer in 0.5 ml dimethylsulfoxide, then adding 1.5 mldichloromethane dropwise. The polymer solution is emulsified in 100 mlof 5% w/v polyvinyl alcohol solution (average molecular weight 25 KDa,88% hydrolyzed) using a homogenizer (Silverson) at a speed ofapproximately 7500 rpm. The resulting dispersion is stirred using amagnetic stirrer for 1 hour. Following this period, the pH is brought to7.0-7.2 by addition of 0.1 N NaOH solution. Stirring is continued for anadditional 2 hours until the methylene chloride is completely evaporatedand the particles hardened. The particles are then isolated bycentrifugation at 4000 rpm (1600 g) for 10 minutes (Sorvall RX-5B). Thesupernatant is discarded and the precipitate washed three times withdistilled water, the dispersion frozen in liquid nitrogen, andlyophilized (Labconco freeze dryer 8) for at least 48 hours. Particlesizing is performed using a Coulter counter. Average particle meandiameters ranged from 100 nm to 14 μm, depending upon processingparameters such as homogenization speed and time. All particles exhibithigh porosity (net mass density less than 0.4 g/cm³). Scanning electronmicrograph photos of the particles showed them to be highly porous.

Porous PLAL-Ala-Lys Particles

100 mg of PLAL-Ala-Lys is completely dissolved in 0.4 mltrifluoroethanol, then 1.0 ml methylene chloride is added dropwise. Thepolymer solution is emulsified in 100 ml of 1% w/v polyvinyl alcoholsolution (average molecular weight 25 KDa, 80% hydrolyzed) using asonicator (Sonic&Materal VC-250) for 15 seconds at an output of 40 W. 2ml of 1% PVA solution is added to the mixture and it is vortexed at thehighest speed for 30 seconds. The mixture is quickly poured into abeaker containing 100 ml 0.3% PVA solution, and stirred for three hoursallowing evaporation of the methylene chloride. Scanning electronmicrograph photos of the particles showed them to be highly porous.

Porous Copolymer Particles

Polymeric porous particles consisting of a blend of PLAL-Lys ad PLGA-PEGwere made. 50 mg of the PLGA-PEG polymer (molecular weight of PEG: 20KDa, 1:2 weight ratio of PEG:PLGA, 75:25 lactide:glycolide) wascompletely dissolved in 1 ml dichloromethane. 3 mg ofpoly(lactide-co-lysine)-polylysine graft copolymer is dissolved in 0.1ml dimethylsulfoxide and mixed with the first polymer solution. 0.2 mlTE buffer, pH 7.6, is emulsified in the polymer solution by probesonication (Sonic&Materal VC-250) for 10 seconds at an output of 40 W.To this first emulsion, 2 ml of distilled water is added and mixed usinga vortex mixer at 4000 rpm for 60 seconds. The resulting dispersion isagitated by using a magnetic stirrer for 3 hours until methylenechloride is completely evaporated and microspheres formed. The spheresare then isolated by centrifugation at 5000 rpm for 30 min. Thesupernatant is discarded, the precipitate washed three times withdistilled water and resuspended in 5 ml of water. The dispersion isfrozen in liquid nitrogen and lyophilized for 48 hours.

Variables which may be manipulated to alter the size distribution of theparticles include: polymer concentration, polymer molecular weight,surfactant type (e.g., PVA, PEG, etc.), surfactant concentration, andmixing intensity. Variables which may be manipulated to alter theporosity of the particles include: polymer concentration, polymermolecular weight, rate of methylene chloride extraction by isopropylalcohol (or another miscible solvent), volume of isopropyl alcoholadded, inclusion of an inner water phase, volume of inner water phase,inclusion of salts or other highly water-soluble molecules in the innerwater phase which leak out of the hardening sphere by osmotic pressure,causing the formation of channels, or pores, in proportion to theirconcentration, and surfactant type and concentration.

By scanning electron microscopy (SEM), the PLAL-Lys-PLGA-PEG particleswere highly porous. The particles had a mean particle diameter of 7μm±3.8 μm. The blend of PLAL-Lys with poly(lactic acid) (PLA) and/orPLGA-PEG copolymers can be adjusted to adjust particle porosity andsize. Additionally, processing parameters such as homogenization speedand time can be adjusted. Neither PLAL, PLA nor PLGA-PEG alone yields aporous structure when prepared by these techniques.

EXAMPLE 3 Rhodamine Isothiocyanate Labeling of PLAL and PLAL-LysParticles

Lysine amine groups on the surface or porous (PLAL-Lys) and nonporous(PLAL) microparticles with similar mean diameters (6-7 μm) and sizedistributions (standard deviations 3-4 μm) were labeled with Rhodamineisothiaocyanate. The mass density of the porous PLAL-Lys particles was0.1 g/cm³ and that of the nonporous PLAL particles was 0.8 g/cm³.

The rhodamine-labeled particles were characterized by confocalmicroscopy. A limited number of lysine functionalities on the surface ofthe solid particle were able to react with rhodamine isothiocyanate, asevidenced by the fluorescent image. In the porous particle, the higherlysine content in the graft copolymer and the porous particle structureresult in a higher level of rhodamine attachment, with rhodamineattachment dispersed throughout the interstices of the porous structure.This also demonstrates that targeting molecules can be attached to theporous particles for interaction with specific receptor sites within thelungs via chemical attachment of appropriate targeting agents to theparticle surface.

EXAMPLE 4 Aerosolization of PLAL and PLAL-Lys Particles

To determine whether large porous particles can escape (mouth, throat,and inhaler) deposition and more efficiently enter the airways and acinithan nonporous particles of similar size, aerosolization and depositionof porous PLAL-Lys (mean diameter 6.3 μm±3.3 μm) or nonporous PLAL (meandiameter 6.9 μm±3.6 μm) particles were examined in vitro using a cascadeimpactor system.

20 mg of the porous or nonporous microparticles were placed in gelatincapsules (Eli Lilly), the capsules loaded into a SPINHALER® dry powderinhaler (DPI) (Fisons), and the DPI activated. Particles wereaerosolized into a Mark I Andersen Impactor (Anderson Samplers, Ga.)from the DPI for 30 seconds at 28.3l/min flow rate. Each plate of theAndersen Impactor was previously coated with Tween 80 by immersing theplates in an acetone solution (5% w/vol) and subsequently evaporatingthe acetone in a oven at 60° for 5 min. After aerosolization anddeposition, particles were collected from each stage of the impactorsystem in separate volumetric flasks by rinsing each stage with NaOHsolution (0.2 N) in order to completely degrade the polymers. Afterincubation at 37° C. for 12 h, the fluorescence of each solution wasmeasured (wavelengths of 554 nm excitation, 574 nm emission).

Particles were determined as nonrespirable (mean aerodynamic diameterexceeding 4.7 .mu.m: impactor estimate) if they deposited on the firstthree stages of the impactor, and respirable (mean aerodynamic diameter4.7 .mu.m or less) if they deposited on subsequent stages. FIG. 1 showsthat less than 10% of the nonporous (PLAL) particles that exit the DPIare respirable. This is consistent with the large size of themicroparticles and their standard mass density. On the other hand,greater than 55% of the porous (PLAL-Lys) particles are respirable, eventhough the geometrical dimensions of the two particle types are almostidentical. The lower mass density of the porous (PLAL-Lys)microparticles is responsible for this improvement in particlepenetration, as discussed further below.

The nonporous (PLAL) particles also inefficiently aerosolize from theDPI; typically, less than 40% of the nonporous particles exited theSpinhaler DPI for the protocol used. The porous (PLAL-Lys) particlesexhibited much more efficient aerosolization (approximately 80% if theporous microparticles typically exited the DPI during aerosolization).

The combined effects of efficient aerosolization and high respirablefraction of aerosolized particle mass means that a far greater fractionof a porous particle powder is likely to deposit in the lungs than of anonporous particle powder.

EXAMPLE 5 In Vivo Aerosolization of PLAL and PLAL-Lys Particles

The penetration of porous and non-porous polymeric PLAL and PLAL-Lysmicroparticles into the lungs was evaluated in and in vivo experimentinvolving the aerosolization of the microparticles into the airways oflive rats.

Male Sprague Dawley rats (150-200 g) were anesthetized using ketamine(90 mg/kg)/xylazine (10 mg/kg). The anesthetized rat was placed ventralside up on a surgical table provided with a temperature controlled padto maintain physiological temperature. The animal was cannulated aboutthe carina with an endotracheal tube connected to a Harvard ventilator.The animal was force ventilated for 20 minutes and 300 ml/min. 50 mg ofporous (PLAL-Lys) or nonporous (PLA) microparticles were introduced intothe endotracheal tube.

Following the period of forced ventilation, the animal was euthanizedand the lungs and trachea were separately washed using bronchoalveolarlavage. A tracheal cannula was inserted, tied into place, and theairways were washed with 10 ml aliquots of HBSS. The lavage procedurewas repeated until a total volume of 30 ml was collected. The lavagefluid was centrifuged (400 g) and the pellets collected and resuspendedin 2 ml of phenol red-free Hanks balanced salt solution (Gibco, GrandIsland, N.Y.) without Ca²+ and Mg²+ (HBSS). 100 ml were removed forparticle counting using a hemacytometer. The remaining solution wasmixed with 10 ml of 0.4 N NaOH. After incubation at 37° C. for 12 h, thefluorescence of each solution was measured (wavelengths of 554 nmexcitation, 574 nm emission).

FIG. 2 is a bar graph showing total particle mass deposited in thetrachea and after the carina (lungs) in rat lungs and upper airwaysfollowing intratracheal aerosolization during forced ventilation. ThePLAL-Lys porous particles had a mean diameter 6.9 μm±4.2 μm. Thenonporous particles PLAL particles had a mean diameter of 6.7 μm±3.2 μm.Percent tracheal porous particle deposition was 54.54±0.77, andnonporous deposition was 76.98±1.95. Percent porous particle depositionin the lungs was 46.75±0.77, and nonporous deposition was 23.02±1.95.

The nonporous (PLAL) particles deposited primarily in the trachea(approximately 79% of all particle mass that entered the trachea). Thisresult is similar to the in vitro performance of the nonporousmicroparticles and is consistent with the relatively large size of thenonporous particles. Approximately 54% of the porous (PLAL-Lys) particlemass deposited in the trachea. Therefore, about half of the porousparticle mass that enters the trachea traverses through the trachea andinto the airways and acini of the rat lungs, demonstrating the effectivepenetration of the porous particles into the lungs.

Following bronchoalveolar lavage, particles remaining in the rat lungswere obtained by careful dissection of the individual lobes of thelungs. The lobes were placed in separate petri dishes containing 5 ml ofHBSS. Each lobe was teased through 60 mesh screen to dissociate thetissue and was then filtered through cotton gauze to remove tissuedebris and connective tissue. The petri dish and gauze were washed withan additional 15 ml of HBSS to maximize microparticle collection. Eachtissue preparation was centrifuged and resuspended in 2 ml of HBSS andthe number of particles counted in a hemacytometer. The particle numbersremaining in the lungs following the bronchoalveolar lavage are shown inFIG. 3. Lobe numbers correspond to: 1) left lung, 2) anterior, 3)median, 4) posterior, 5) postcaval. A considerably greater number ofporous PLAL-Lys particles enters every lobe of the lungs than thenonporous PLAL particles, even though the geometrical dimensions of thetwo types of particles are essentially the same. These results reflectboth the efficiency of porous particle aerosolization and the propensityof the porous particles to escape deposition prior to the carina orfirst bifurcation.

Modifications and variations of the present invention will be obvious tothose skilled in the art from the foregoing detailed description. Suchmodifications and variations are intended to come within the scope ofthe following claims.

1. Therapeutic particles suitable for aerosolization in a dry powderinhaler (DPI) comprising, a therapeutic agent and a pharmaceuticallyacceptable carrier, wherein the particles are porous, have a tap densityless than about 0.4 g/cm³ and wherein upon aerosolization, about 80% ofthe particles exit the DPI and at least 55% of the particles exiting theDPI have an aerodynamic diameter of less than about 4.7 μm as measuredby a cascade impactor for 30 seconds at 28.3 l/min flow rate.
 2. Theparticles of claim 1, wherein the particles have a tap density less thanabout 0.1 g/cm³.
 3. The particles of claim 1, wherein the particles havea mass mean diameter between 5 μm and 30 μm.
 4. The particles of claim1, wherein the pharmaceutically acceptable carrier comprises a poreforming material.
 5. The particles of claim 1, wherein thepharmaceutically acceptable carrier is a surfactant.
 6. The particles ofclaim 1, wherein the agent is selected from the group consisting ofproteins, peptides, polysaccharides, lipids, nucleic acids andcombinations thereof.
 7. The particles of claim 1, wherein the agent isselected from the group consisting of antibodies, antigens, antibiotics,antivirals, hormones, vasoactive agents, neuroactive agents,anticoagulants, immunomodulating agents, cytotoxic agents, ribozymes,antisense agents and genes.
 8. The particles of claim 1, for use inlocal inhalation therapy.
 9. The particles of claim 8, wherein the agentis an agent for the treatment of asthma, emphysema, or cystic fibrosis.10. The particles of claim 1, for use in systemic inhalation therapy.11. The particles of claim 10, wherein the agent is insulin.
 12. Amethod of enhancing the aerosolization efficiency of therapeuticparticles administered to a patient by a dry powder inhaler comprisingthe steps of: a) providing particles suitable for aerosolization in adry powder inhaler (DPI) wherein the particles are porous, have a tapdensity less than about 0.4 g/cm³ and comprise a therapeutic agent and apharmaceutically acceptable carrier, and wherein upon aerosolization,about 80% of the particles exit the DPI and at least 55% of theparticles exiting the DPI have an aerodynamic diameter of less thanabout 4.7 μm as measured by a cascade impactor for 30 seconds at 28.3l/min flow rate; and b) aerosolizing the particles by activating the DPIsuch that the particles exit the DPI and are administered to thepatient.
 13. The method of claim 12, wherein the particles have a tapdensity less than about 0.1 g/cm³.
 14. The method of claim 12, whereinthe particles have a mass mean diameter between 5 μm and 30 μm.
 15. Themethod of claim 12, wherein the pharmaceutically acceptable carriercomprises a pore forming material.
 16. The method of claim 12, whereinthe pharmaceutically acceptable carrier is a surfactant.
 17. The methodof claim 12, wherein the agent is selected from the group consisting ofproteins, peptides, polysaccharides, lipids, nucleic acids andcombinations thereof.
 18. The method of claim 12, wherein the agent isselected from the group consisting of antibodies, antigens, antibiotics,antivirals, hormones, vasoactive agents, neuroactive agents,anticoagulants, immunomodulating agents, cytotoxic agents, ribozymes,antisense agents and genes.
 19. The method of claim 12, for use in localinhalation therapy.
 20. The method of claim 19, wherein the agent is anagent for the treatment of asthma, emphysema, or cystic fibrosis. 21.The method of claim 12, for use in systemic inhalation therapy.
 22. Themethod of claim 21, wherein the agent is insulin.